Compositions and methods for the treatment of plaques and tangles in humans and animals

ABSTRACT

Composition and methods for treating brain “plaques” and “tangles” in a patient or an animal wherein the method comprises administration of a therapeutically effective amount of a composition comprising Uncaria  tomentosa  extract and an oolong tea extract.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.15/669,443, filed Aug. 4, 2017, which is a Continuation of U.S. patentapplication Ser. No. 15/053,572, filed Feb. 25, 2016, which claimspriority to U.S. Provisional Application 62/126,026, filed Feb. 27,2015, and U.S. Provisional Application 62/170,822, filed Jun. 4, 2015,each of which are incorporated by reference in their entireties.

TECHNICAL FIELD

The invention relates to blended compositions of plant extracts ofUncaria tomentosa and Oolong tea and methods for treating “plaques andtangles” that accumulate in the aging brain in amyloidosis andtauopathies in humans and animals (i.e. such as aged dogs and cats). Inaddition, the invention relates to the development of a nutraceuticalblended composition consisting of a combination of a plant extract ofUncaria tomentosa and a specific Oolong tea, to prevent and treattraumatic brain injury (TBI), concussions (as observed in most athletesand the military/soldiers), single and repeated blows to the head, andchronic traumatic encephalopathy (CTE).

BACKGROUND Amyloidosis and the Accumulation of Beta-Amyloid “Plaques inthe Brain of a Variety of Disorders

Alzheimer's disease is characterized by the accumulation of a 39-43amino acid peptide termed the beta-amyloid protein or Aβ, in afibrillary form, existing as extracellular amyloid plaques and asamyloid within the walls of cerebral blood vessels. Fibrillar Aβ amyloiddeposition in Alzheimer's disease is believed to be detrimental to thepatient and eventually leads to toxicity and neuronal cell death, acharacteristic hallmark of Alzheimer's disease. Accumulating evidenceimplicates amyloid, and more specifically, the formation, deposition,accumulation and/or persistence of Aβ fibrils, as a major causativefactor of Alzheimer's disease pathogenesis. In addition, besidesAlzheimer's disease, a number of other amyloid diseases involveformation, deposition, accumulation and persistence of Aβ fibrils,including Down's syndrome, disorders involving congophilic angiopathy,such as but not limited to, hereditary cerebral hemorrhage of the Dutchtype, inclusion body myositosis, dementia pugilistica, cerebralβ-amyloid angiopathy, dementia associated with progressive supranuclearpalsy, dementia associated with cortical basal degeneration and mildcognitive impairment.

The amyloid diseases (amyloidosis) are classified according to the typeof amyloid protein as well as the underlying disease. Amyloid diseaseshave a number of common characteristics including each amyloidconsisting of a unique type of amyloid protein. The amyloid diseasesinclude, but are not limited to, the amyloid associated with Alzheimer'sdisease, Down's syndrome, Canine Dysfunction syndrome (CDS), CanineCognitive Dysfunction (CCD), as seen in aged animals such as dogs andcats, hereditary cerebral hemorrhage with amyloidosis of the Dutch type,dementia pugilistica, inclusion body myositosis (Askanas et al., Ann.Neurol. 43:521-560, 1993) and mild cognitive impairment (where thespecific amyloid is referred to as beta-amyloid protein or Aβ), theamyloid associated with chronic inflammation, various forms ofmalignancy and Familial Mediterranean Fever (where the specific amyloidis referred to as AA amyloid or inflammation-associated amyloidosis),the amyloid associated with multiple myeloma and other B-cell dyscrasias(where the specific amyloid is referred to as AL amyloid), the amyloidassociated with type 2 diabetes (where the specific amyloid is referredto as amylin or islet amyloid polypeptide or IAPP), the amyloidassociated with the prion diseases including Creutzfeld-Jakob disease,Gerstamann-Straussler syndrome, kuru and animal scrapie (where thespecific amyloid is referred to as PrP amyloid), the amyloid associatedwith long-term hemodialysis and carpal tunnel syndrome (where thespecific amyloid is referred to as α2-microglobulin amyloid), theamyloid associated with senile cardiac amyloidosis and FamilialAmyloidotic Polyneuropathy (where the specific amyloid is referred to astransthyretin or prealbumin), and the amyloid associated with endocrinetumors such as medullary carcinoma of the thyroid (where the specificamyloid is referred to as variants of procalcitonin). In addition, theα-synuclein protein which forms amyloid-like fibrils, and is Congo redand Thioflavin S positive (specific stains used to detect amyloidfibrillary deposits), is found as part of Lewy bodies in the brains ofpatients with Parkinson's disease, Lewy body disease (Lewy in Handbuchder Neurologie, M. Lewandowski, ed., Springer, Berlin pp. 920-933, 1912;Pollanen et al, J. Neuropath. Exp. Neurol. 52:183-191, 1993; Spillantiniet al, Proc. Natl. Acad. Sci. USA 95:6469-6473, 1998; Arai et al,Neurosc. Lett. 259:83-86, 1999), multiple system atrophy (Wakabayashi etal, Acta Neuropath. 96:445-452, 1998), dementia with Lewy bodies, andthe Lewy body variant of Alzheimer's disease. For purposes of thisdisclosure, Parkinson's disease, due to the fact that fibrils develop inthe brains of patients with this disease (which are Congo red andThioflavin S positive, and which contain predominant beta-pleated sheetsecondary structure), is now regarded as a disease that also displaysthe characteristics of an amyloid-disease.

Amyloid as a Therapeutic Target for Alzheimer's Disease

Alzheimer's disease is characterized by the deposition and accumulationof a 39-43 amino acid peptide termed the beta-amyloid protein, Aβ orβ/A4 (Glenner and Wong, Biochem. Biophys. Res. Comm. 120:885-890, 1984;Masters et al, Proc. Natl. Acad. Sci. USA 82:4245-4249, 1985; Husby etal, Bull. WHO 71:105-108, 1993). A13 is derived by protease cleavagefrom larger precursor proteins termed β-amyloid precursor proteins(APPs) of which there are several alternatively spliced variants. Themost abundant forms of the APPs include proteins consisting of 696, 751and 770 amino acids (Tanzi et al, Nature 31:528-530, 19980.

The small Aβ peptide is a major component that makes up the amyloiddeposits or “plaques” in the brains of patients with Alzheimer'sdisease. In addition, Alzheimer's disease is characterized by thepresence of numerous neurofibrillary “tangles”, consisting of pairedhelical filaments which abnormally accumulate in the neuronal cytoplasm(Grundke-Iqbal et al, Proc. Natl. Acad. Sci. USA 83:4913-4917, 1986;Kosik et al, Proc. Natl. Acad. Sci. USA 83:4044-4048, 1986; Lee et al,Science 251:675-678, 1991). The pathological hallmark of Alzheimer'sdisease is therefore the presence of both “plaques” and “tangles”, withamyloid being deposited in the central core of the plaques. The othermajor type of lesion found in the Alzheimer's disease brain is theaccumulation of amyloid in the walls of blood vessels, both within thebrain parenchyma and in the walls of meningeal vessels that lie outsidethe brain. The amyloid deposits localized to the walls of blood vesselsare referred to as cerebrovascular amyloid or congophilic angiopathy(Mandybur, J. Neuropath. Exp. Neurol. 45:79-90, 1986; Pardridge et al.,J. Neurochem. 49:1394-1401, 1987).

For many years there has been an ongoing scientific debate as to theimportance of “amyloid” in Alzheimer's disease, and whether the“plaques” and “tangles” characteristic of this disease were a cause ormerely a consequence of the disease. Within the last few years, studiesnow indicate that amyloid is indeed a causative factor for Alzheimer'sdisease and should not be regarded as merely an innocent bystander. TheAlzheimer's Aβ protein in cell culture has been shown to causedegeneration of nerve cells within short periods of time (Pike et al,Br. Res. 563:311-314, 1991; J. Neurochem. 64:253-265, 1995). Studiesshow that it is the fibrillary structure (consisting of a predominantβ-pleated sheet secondary structure) characteristic of all amyloids thatis responsible for the neurotoxic effects. Aβ has also been found to beneurotoxic in slice cultures of hippocampus (Harrigan et al, Neurobiol.Aging 16:779-789, 1995) and to induce nerve cell death in transgenicmice (Games et al, Nature 373:523-527, 1995; Hsiao et al, Science272:99-102, 1996). Injection of the Alzheimer's Aβ into rat brain alsocauses memory impairment and neuronal dysfunction (Flood et al, Proc.Natl. Acad. Sci. USA 88:3363-3366, 1991; Br. Res. 663:271-276, 1994).

Probably, the most convincing evidence that Aβ amyloid is directlyinvolved in the pathogenesis of Alzheimer's disease comes from geneticstudies. It was discovered that the production of Aβ can result frommutations in the gene encoding its precursor, β-amyloid precursorprotein (Van Broeckhoven et al, Science 248:1120-1122, 1990; Murrell etal, Science 254:97-99, 1991; Haass et al, Nature Med. 1: 1291-1296,1995). The identification of mutations in the beta-amyloid precursorprotein gene that cause early onset familial Alzheimer's disease is thestrongest argument that amyloid is central to the pathogenic processunderlying this diseases. Several reported disease-causing mutationshave been discovered which demonstrate the importance of AP in causingfamilial Alzheimer's disease (reviewed in Hardy, Nature Genet.1:223-234, 1992). All of these studies suggest that providing a therapy,drug or supplement to reduce, eliminate and/or prevent fibrillary Aβformation, deposition, accumulation and/or persistence in the brains ofhumans and animals, well serve as an effective therapeutic.

The Accumulation of “Plaques and Tangles” in the Aging Human and AnimalBrain

The human brain is the most complex organ in the universe. It weighsonly 3 pounds, or about 2% of body weight. Yet is uses 20-30% of thecalories consumed, 20% of the oxygen breathed, and 25% of the blood flowin the body; it consists of 85% water (Daniel G. Amen, M. D. 12prescriptions for creating a brain healthy life. Source:www.amenclinics.com/cybcyb/12-prescriptions-for-creating-a-brain-healthy-life/).There are approximately 100 billion nerve cells (i.e. neurons) in thebrain, and up to a quadrillion connections called synapses (ibid). Thehuman brain as it ages, loses about 85,000 cortical neurons per day, orabout one every second (Deepak Chopra, M. D. and Rudolph Tanzi, Ph.D.Super Brain. Unleashing the Explosive Power of Your Mind to MaximizeHealth, Happiness, and Spiritual Well Being.See///www.chopra.com/super-brain-by-deepak-chopra-rudolph-tanzi.) As thebrain ages, starting in the 20's, there is a slow but deliberateaccumulation of two neurotoxic proteins. The first is the brainaccumulation of an insoluble (aggregated) specific neurotoxic proteinknown as the “beta-amyloid protein” or AP. Beta-amyloid protein depositsin the form of “plaques” (looking like “meatballs” in the brain under amicroscope), have been shown to be instrumental in killing healthyneurons that lead to a decline in hippocampus-dependent memory andcognition. Dr. Alan Snow and co-inventors developed patented methods toproduce “plaques in a test-tube” (identical to what is seen in the humanbrain) and used these methods to screen for and identify natural“plaque-reducing” nutraceutical ingredients (U.S. Pat. No. 7,148,001,which is incorporated herein by reference in its entirety).

The second neurotoxic protein that accumulates in the aging brain isknown as the “tau protein” and forms twisted paired helical filamentsknown as “tangles.” Neurofibrillary tangles accumulate inside neuronsthat causes them to die, and look like “dried spaghetti strands” under amicroscope. Dr. Snow's laboratories developed proprietary methods toform “tangles” in a test-tube, and then used these assays to identify“tangle-inhibiting” nutraceutical ingredients (see examples below).Thus, in the aging brain, both “plaques and tangles” accumulate, causingneurons to die; connections between nerve cells (called synapses) todisintegrate; and memory and cognition to progressively decline.Compounds or agents able to disaggregate and reduce the accumulation of“plaques and tangles” have been shown to lead to memory improvement anda reduction in memory decline (Karinoski et al. Suppression of amyloiddeposition leads to long-term reductions in Alzheimer's pathologies inTg2576 mice. J. Neurosc. 29:4964-4971, 2009; Vellas et al. Long-termfollow-up of patients immunized with AN1792: Reduced functional declinein antibody responders. Current Alz. Res. 6:144-151, 2009; Morgan et al.Aβ peptide vaccination prevents memory loss in an animal model ofAlzheimer's disease. Nature 408:982-985, 2000; Chen et al. A learningdeficit related to age and β-amyloid plaques in a mouse model ofAlzheimer's disease. Nature 408:975-979, 2000; Janus et al. Aβ peptideimmunization reduces behavioral impairment and plaques in a model ofAlzheimer's disease. Nature 408:979-985, 2000; Schenk et al.Immunization with amyloid-β attenuates Alzheimer-disease like pathologyin PDAPP mouse. Nature 400:173-177, 1999; Yanamandra et al. Anti-tauantibodies that block tau aggregate seeding in vitro markedly decreasespathology and improves cognition in vivo. Neuron 80:402-414, 2013;Dumont et al. Bezafibrate administration improves behavioral deficitsand tau pathology in P3015 mice. Human Molecular Genetics 21:5091-5105,2012; Oddo et al. Reduction of soluble Abeta and tau, but not solubleAbeta alone, ameliorates cognitive decline in transgenic mice withplaques and tangles. J. Biol. Chem. 281:39413-39423, 2006; Santacruz etal. Tau suppression in a neurodegenerative mouse model improves memoryfunction. Science 309:476-481, 2005.)

The only difference between an aging brain that could lead toage-associated memory impairment (AAMI), then to mild-cognitiveimpairment (MCI), and potentially to Alzheimer's disease, and a brainthat does not, is the number of “plaques and tangles” in the brain.Alzheimer's diseased brains are loaded with tens to hundreds ofthousands of “plaques and tangles,” per square millimeter, causing amarked increase in the death of neurons, leading to a loss of synapses(connections between neurons), and concurrent memory loss and cognitivedecline.

Therefore, beta-amyloid and tau are two key proteins in the aging brainthat accumulate as insoluble “plaques and tangles” that have been shownto be directly linked to memory loss and cognitive decline. There iscurrently no pharmaceutical drug that has been approved for reducing andremoving both beta-amyloid protein “plaques” and tau protein-containing“tangles” in the brain.

The Accumulation of “Plaques” in the Aging Dog and Cat Brain

Dogs and cats also accumulate “plaques” (and, to a lesser extent,“tangles”) in their brains as they age that are believed to contributeto memory decline and cognitive impairment. The same beta-amyloidprotein (i.e. “plaques”) and tau protein (“tangles”) that accumulate inthe human brain also accumulate in aged dogs (Papoiannou et al,Immunohistochemical investigation of the brain of aged dogs. I.Detection of neurofibrillary tangles and of 4-hydroxynonenal protein, anoxidative damage product, in senile plaques. Amyloid 8:11-21, 2001;Uchida et al, Amyloid angiopathy with cerebral hemorrhage and senileplaque in aged dogs. Nihon Juigaku Zasshi 52: 605-11, 1990) and cats(Gunn-Moore et al, Cognitive dysfunction and the neurobiology of ageingin cats. J Small Anim. Pract. 48: 546-53, 2007; Nakamura et al. Senileplaques in very aged cats. Acta Neuropath. 91: 437-9, 1996).

Canine Cognitive Dysfunction (CCD) (also known as Cognitive DysfunctionSyndrome or CDS) is a disease prevalent in dogs (and cats) that exhibitsymptoms of dementia or Alzheimer's disease as seen in humans. CCDcreates pathological changes in the brain that slow the mentalfunctioning of dogs (and cats) resulting in loss of memory, motorfunction and learned behaviors from training early in life. In the dog'sand cat's brain, the culprit is again is the beta-amyloid protein or Aβthat forms “plaques” in the brain. As the dog ages, more and more“plaques” accumulate and nerve cells die. Although the initial symptomsof the disorders are mild, they gradually worsen over time, also knownas “cognitive decline”. Amyloid “plaques” occur in aged dogs at aboutfive to seven years of age, and in cats of about ten years of age (whichis proportional to their average lifespan of 15-20 years). In fact,clinical signs of cognitive dysfunction syndrome are found in 50% ofdogs over the age of 11, and by the age of 15, 68% of dogs display atleast one sign. Dogs will often find themselves confused in familiarplaces of the home, spending long periods of time in one area of thehome, not responding to calls or commands, and experiencing abnormalsleeping patterns.

Beta-amyloid protein containing “plaques” also have been identified inthe brains of other higher mammals including monkeys, bears, camels, andhorses. (Nakamura et al, Histopathological studies of senile plaques andcerebral amyloidosis in cynomolgus monkeys. J Med Primatol. 27: 244-52,1998; Capucchio et al. studies. J Comp Pathol 142: 61-73, 2010; Nakamuraet al, Senile plaques in an aged two-humped (Bactrian) camel (Camelusbactrianus), Acta Neuropathol 90: 415-8, 1995; Uchida et al, Senileplaques and other senile changes in the brain of an aged American blackbear, Vet. Pathol. 32: 412-4, 1995).

Tauopathies and “Tangles”

Tau was discovered in the mid-1970s as a microtubule associated protein(Weingarten, 1975). Besides being a stabilizer of microtubules inneurons and other cells, it has since been found to play important rolesin cell differentiation, polarization and axonal transport. Normal tauis a soluble protein bound to microtubules, but in a series ofneurodegenerative diseases, now known as tauopathies, tau accumulates asa pathogenic insoluble, fibrillar protein. These tau inclusions appearto modulate the severity of dementia and clinical features of theseneurodegenerative diseases. Tauopathies include diseases such asAlzheimer's disease, frontotemporal lobar degeneration with tauinclusions (FTLD-tau) such as Pick's disease, progressive supranuclearpalsy, and corticobasal degeneration, agyrophillic grain disease, someprion diseases, amyotrophic lateral sclerosis/parkinsonism-dementiacomplex, chronic traumatic encephalopathy, and some genetic forms ofParkinson's disease (V. M. Lee et al., Ann. Rev. Neurosci. 24:1121-1159, 2001; B. Omalu et al., Neurosurgery 69(1):173-83, 2011; A.Rajput et al., Neurology 67: 1506-1508, 2006; G. Santpere and I. Ferrer,Acta Neuropathol. 117: 227-246, 2009).

One of the most notable effects of increasing fibrillar tau in the brainis the gradual deterioration of short term memory; that is, the abilityto recall immediately those memories only recently stored (P.Giannakopoulos et al., Neurology 60: 1495-1500, 2003). As there is notreatment for these disorders, it is important to find a novel inventionthat could target this pathogenic protein and improve memory deficits.

“Tangles” Accumulate in Brain in Traumatic Brain Injury (TBI),Concussions, Head Trauma and Chronic Traumatic Encephalopathy (CTE)

Brain “tangles” consisting of tau protein also accumulate progressivelyin the brain following blows to the head and include concussions, headinjury, post-traumatic stress disorder (PTSD), and blast-inducedtraumatic brain injury (seen in soldiers and military personnel who havetraumatic head injuries induced by a single blast). The movie“concussion” and the NFL Players Association all discuss thedementia-type behavior that has been seen in athletes following repeatedconcussions and/or blows to the head (known as traumatic brain injury orTBI). Loss of consciousness is a clinical hallmark of concussion but isnot required to make the diagnosis. Other symptoms include confusion,disorientation, unsteadiness, dizziness, headache, and visualdisturbances.

The long-term consequences to traumatic brain injury is referred to asChronic Traumatic Encephalopathy (CTE), which is form of tauopathy (i.e.tau protein containing “tangles” in the brain). CTE is a progressivedegenerative disease found in people who have suffered repeated braintrauma including sub-concussive hits to the head that do not causeimmediate symptoms. The disease was previously called dementiapugilistica (DP), i.e. “punch-drunk” as it was initially found in thosewith a history of boxing. CTE has now been found in the brains ofprofessional athletes including NFL athletes who play football, athletesprone to head injury including those that play ice hockey, rugby,skiing, skateboarding, stunt performing, bull riding, rodeo, and allother contact sports where participants experience repeated braintrauma. Individuals with CTE show many signs of dementia such as memoryloss, aggression, confusion, and depression, which may appear years ormany decades after the trauma. In September 2015, researchers with theDepartment of Veterans Affairs and Boston University announced they hadidentified CTE in 96% of NFL football players that they had examined andin 79% of all football players (Jason Breslow, New: 87 deceased NFLplayers test positive for brain disease. Frontline Jan. 9, 2016).

The neuropathology under a microscope is clear—there is primarily anaccumulation of “tangles” that consist of tau protein, similar to the“tangles” seen in the brains of Alzheimer's disease patients. There isalso some beta-amyloid protein deposition (i.e. “plaques”), but this isusually uncommon and less of a feature then the “tangle” accumulation inbrain. These findings suggest that blows to the head can lead to nearimmediate brain “tangle” accumulation that then leads to dementia-likesymptoms including memory loss and cognitive decline. Identification ofa nutraceutical that can help in the reduction and/or clearance of brain“tangles” would be an extraordinary supplement to take every day by allkinds of athletes, NFL players, the military and its soldiers.

Disclosures

An object of the present invention is to provide the combination ofplant extracts from Uncaria tomentosa and an oolong tea extract for thedevelopment of a cognition and memory supplement, to prevent, reduceand/or clear brain “plaques and tangles” in humans. This invention ispredicated on the surprising discovery that various plant extracts, evenfrom the same plant source have differing effectiveness for dissolvingplaques and tangles, and that oolong tea extract, in particular LOTE, incombination with Uncaria tomentosa extract has a surprising andunexpected activity compared other plant extracts or with each extractalone.

Another object of the present invention is to provide the combination ofplant extracts from Uncaria tomentosa and an oolong tea extract toproduce a pet food supplement for aged dogs that reduce brain amyloid“plaques” and improve cognition, memory, short-term memory, focus andconcentration.

Another object of the present invention is to provide the combination ofplant extracts from Uncaria tomentosa and an oolong tea extract toproduce a pet food supplement for aged cats that reduce brain amyloid“plaques” and improve cognition, memory, short-term memory, focus andconcentration.

Although some health care providers have suggested that Uncariatomentosa may be used to treat a variety of ailments, nowhere has therebeen any use, or suggestion of use, of this compound for the treatmentof tau fibril formation, deposition, accumulation and/or persistence,such as that which occurs in the tauopathies. Furthermore, nowhere is itsuggested that certain other compounds might have synergistic orsupplemental efficacy in combination with Uncaria tomentosa in treatingtauopathies to prevent and/or treat, for example, traumatic brain injury(TBI), concussions (as observed in most athletes and themilitary/soldiers), post-traumatic stress disorders, head trauma, blowsto the head (either single or repeated), and chronic traumaticencephalopathy (CTE).

Another object of the present invention is to provide the combination ofplant extracts from Uncaria tomentosa and an oolong tea extract toproduce an effective preventer and/or reducer of brain “tangles,” suchas those found in people with traumatic brain injury (TBI), concussions(as observed in most athletes and the military/soldiers), post-traumaticstress disorders, head trauma, blows to the head (either single orrepeated), and chronic traumatic encephalopathy (CTE).

The present invention clearly demonstrates the effectiveness of plantextracts of Uncaria tomentosa (cat's claw) in specific combination withan Oolong tea plant extract for 1) inhibition of tau fibril formation(important for patients in early- to mid-stage tauopathy), 2) inhibitionof tau fibril growth (important for patients in early- to mid-stagetauopathy), and 3) causing the dissolution/disruption of preformed taufibrils (important for late-stage tauopathy).

An object of the present invention is to use the inner bark and/or rootsfrom Uncaria tomentosa (also referred to as Una de Gato or Cat's claw)for the treatment/inhibition of tau deposition, accumulation and/orpersistence in tauopathies, in conjunction with an oolong tea extract,disclosed below, to achieve a beneficial therapeutic effect. Uncariatomentosa or Cat's claw is also referred to as, but not limited to,Paraguayo, Garabato, Garbato casha, Tambor huasca, Una de gavilan,Hawk's claw, Nail of Cat, and Nail of Cat Schuler.

Another object of the present invention is to provide the use of Uncariatomentosa with an oolong tea extract (regardless of commercial sourceand regardless of final form for consumption by humans, i.e. pills,tablets, caplets, soft and hard gelatin capsules, lozenges, sachets,cachets, vegicaps, liquid drops, elixirs, suspensions, emulsions,solutions, syrups, tea bags, aerosols (as a solid or in a liquidmedium), suppositories, sterile injectable solutions, sterile packagedpowders, bark bundles and/or bark powder) for inhibition of tau fibrilformation, deposition, accumulation, and/or persistence, regardless ofits clinical setting.

These and such other objects of the invention as will become evidentfrom the disclosure below are met by the invention disclosed herein.

Applications

Application of the invention provides for use of plant extracts fromUncaria tomentosa and a specific oolong tea extract to benefits ageddogs and cats, who develop brain “plaques” (and to a lesser extent,“tangles”), and have dementia as defined in Canine Cognitive Dysfunction(CCD).

We have earlier discovered and disclosed a naturally occurring plantproduct, the inner bark and/or roots from the plant Uncaria tomentosa,or Cat's Claw, that we call PTI-00703°, in WIPO Internationalpublication number WO98/51302 entitled ‘Composition and Methods forTreating Alzheimer's Disease and other Amyloidoses’ dated Nov. 19, 1998,which is incorporated herein by reference in its entirety. As disclosedtherein, this plant compound alone has surprising efficacy in disruptingand/or dissolving amyloid deposits and other accumulations, and isbelieved to be a potent inhibitor of amyloid formation in Alzheimer'sdisease, Type II Diabetes, and other amyloidoses.

However, our data now demonstrates that the formulation of PT1-00703with a specific oolong tea extract (referred to as TOTE′) disclosedherein has surprising and hitherto unsuspected potent efficacy intreating Tauopathies and diseases in which “tangles” are found in thebrain (such as in TBI, CTE in humans with concussions, head injuryand/or head trauma), and diseases that contain an overabundance of“plaques in brain” such as in aged dogs and cats (in Canine CognitiveDysfunction).

The invention relates to the use of mixed compositions including one ormore of the following: oolong tea extract and Uncaria tomentosa extractPTI00703® referred to as ‘703’ or ‘PTI-703’ for the therapeuticintervention of amyloidosis. Use of a mixed composition of an oolong teaextract, and/or PTI00703®, contained within different commercialpreparations show unexpected effects on the inhibition of amyloid fibrilformation and disaggregation of amyloid fibrils.

Application of the invention to these needs is especially beneficial inthat the invention is the only system that effectively provides for useof extracts from the inner bark and root parts of Uncaria tomentosa,together with an oolong tea extract, to benefit human patients withTauopathies, amyloid diseases, “plaques and/or tangles” in the brain,and those with traumatic brain injury (TBI), concussions (as observed inmost athletes and the military/soldiers), post-traumatic stressdisorders, head trauma, blows to the head (either single or repeated),and chronic traumatic encephalopathy (CTE).

Application of the invention to these needs is especially beneficial inthat the invention is the only system that effectively provides for useof extracts from the inner bark and root parts of Uncaria tomentosa,together with an Oolong tea extract, to benefit human patients withtauopathies, due to the newly discovered ability of Uncaria tomentosa incombination with an oolong tea extract, to effectively inhibit taufibril formation, inhibit tau fibril growth, inhibit fibrillartau-proteoglycan interactions, fibrillar tau-glycosaminoglycaninteractions, and cause dissolution and/or disruption of preformed taufibrils.

Over 25 extracts of teas, along with extracts of coffee and yerba mate,were screened for amyloid fibril aggregation inhibition anddisaggregation in vitro using a variety of assays. One lead oolong teaextract was selected and tested in combination with PTI00703®. It wasfound that the combination of this specific lead oolong tea extract(LOTE) and PTI-00703® inhibited amyloid fibril formation more than thetea extract or PTI-00703® alone, and the combination of LOTE andPTI-00703® also had the ability to rapidly disaggregate (within 15minutes) preformed amyloid fibrils.

The invention also relates to the use of mixed compositions includingone or more of the following: oolong tea extract and Uncaria tomentosaextract PTI-00703 referred to as ‘703’ or ‘PTI-703’ for the therapeuticintervention of tauopathies. Use of a mixed composition of oolong teaextract, and/or PTI-00703, contained within different commercialpreparations show unexpected effects on the inhibition of tau fibrilformation and disaggregation of tau fibrils.

Over 25 extracts of teas along with extracts of coffee and yerba matewere screened for tau fibril aggregation inhibition and disaggregationin vitro. One lead oolong tea extract was selected and tested incombination with PTI-00703®. It was found that the combination of thisspecific lead oolong tea extract (LOTE) and PTI-00703® inhibited taufibril formation more than the tea extract or PTI-00703® alone, and alsohas the ability to rapidly disaggregate preformed tau fibrils.

It was also found that the combination of this specific lead oolong teaextract (LOTE) and PTI-00703® inhibited beta-amyloid protein fibrilformation more than the tea extract or PTI-00703® alone, and also hasthe ability to rapidly disaggregate preformed amyloid fibrils.

A preferred pharmacological agent preferably has a therapeuticallyeffective amount of Uncaria tomentosa in a dosage in the range of fromabout 10 to about 1,000 mg/kg of body weight of the patient, and morepreferably in the range of from about 10 to about 100 mg/kg of bodyweight of the patient.

The compositions preferably have a therapeutically effective amount ofthe mixed composition lead oolong tea extract (LOTE) and PTI-00703®, ina dosage in the range of from about 0.1 to about 500 mg/kg of bodyweight of the patient, and more preferably in the range from about 1.0to about 100 mg/kg of body weight of the patient.

Preferred pharmaceutical agents may also have a pharmaceuticallyacceptable carrier, diluent or excipient. The pharmaceutical agentpreferably has a fibril inhibitory activity or efficacy greater than50%.

The plant matter is preferably comprised of commercially obtained pills,tablets, caplets, soft and hard gelatin capsules, lozenges, sachets,cachets, vegicaps, liquid drops, elixirs, suspensions, emulsions,solutions, syrups, tea bags, aerosols (as a solid or in a liquidmedium), suppositories, sterile injectable solutions, sterile packagedpowders, bark bundles and/or bark powder, which contain Uncariatomentosa, extracts or derivatives thereof (and may be taken fromcommercially available gelatin-coated capsules which contain driedpowder of Uncaria tomentosa, extracts or derivatives thereof), incombination with a specific oolong tea extract.

An object of the present invention is to use the inner bark and/or rootsfrom Uncaria tomentosa (also referred to as Una de Gato or Cat's claw)for the treatment/inhibition of amyloid deposition, accumulation and/orpersistence in amyloidosis, in conjunction with an oolong tea extract,disclosed below, to achieve a beneficial therapeutic effect. Uncariatomentosa or cat's claw is also referred to as, but not limited to,Paraguiayo, Garabato, Garbato casha, Tambor huasca, Una de gavilan,Hawk's claw, Nail of Cat, and Nail of Cat Schuler.

Another object of the present invention is to provide the use of Uncariatomentosa with an oolong tea extract (regardless of commercial sourceand regardless of final form for consumption by humans, i.e. pills,tablets, caplets, soft and hard gelatin capsules, lozenges, sachets,cachets, vegicaps, liquid drops, elixirs, suspensions, emulsions,solutions, syrups, tea bags, beverage preparations, aerosals, as a solidor in a liquid medium, suppositories, sterile injectable solutions,sterile packaged powders, bark bundles and/or bark powder, forinhibition of amyloid fibril formation, deposition, accumulation and/orpersistence, regardless of its clinical setting.

Application of the invention to these needs is especially beneficial inthat the invention is the only system that effectively provides the useof extracts from the inner bark and root parts of Uncaria tomentosa,together with a specific Oolong tea extract, to benefit human patientswith amyloidosis, and aged mammals (such as dogs and cats that developamyloid “plaques” in the brain as they age), due to the newly discoveredability of Uncaria tomentosa in combination with an Oolong tea extract,to most remarkably and effectively inhibit amyloid fibril formation,inhibit amyloid fibril growth, and cause dissolution and/or disruptionof preformed amyloid fibrils.

A method is also disclosed for treating tauopathy in a patient,comprising the step of administering to the patient a therapeuticallyeffective amount of plant matter from a plant of the genus Uncaria,species tomentosa, in combination with an Oolong tea extract. The plantmatter is preferably administered orally such as in oral capsules,beverage preparations, or any other method, or by aerosol spray or in aparenterally injectable or infusible form.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing will be provided by the Office upon request and payment ofthe necessary fee.

FIG. 1 A graph of Aβ 1-42 aggregation measured by Thioflavin Tfluorometry and Congo red binding after incubation with lead Oolong teaextract (LOTE) and PTI-00703®

FIG. 2A-C Representative images of Congo red, Thioflavin S, and electronmicrographs of Aβ fibrils+/−LOTE and PTI-00703®

FIG. 3 A graph of Aβ 1-40 aggregation measured by Thioflavin Tfluorometry and Congo red binding after incubation with LOTE andPTI-00703®

FIG. 4 Circular dichroism spectra of Aβ 1-40 after treatment withincreasing concentrations of LOTE and PTI-00703®

FIG. 5A-G Data from various internal tests to characterize TauRD fibrilsfor screening of tau aggregation inhibitors.

FIG. 6 A graph of tau aggregation measured by Thioflavin S fluorometryafter incubation with tea extracts.

FIG. 7 A graph of tau aggregation measured by Thioflavin S fluorometryafter incubation with LOTE and PTI-00703.

FIG. 8 A graph of tau secondary structure measured by circular dichroismspectroscopy after treatment with LOTE.

FIG. 9 Electron micrographs of tau fibril formation with PTI-00703 andLOTE.

FIG. 10 Electron micrographs of preformed tau fibrils treated withPTI-00703 and LOTE.

DETAILED DESCRIPTION

A “therapeutically effective amount” in general means the amount that,when administered to a subject or animal for treating a disease, issufficient to affect the desired degree of treatment for the disease. A“therapeutically effective amount” or a “therapeutically effectivedosage” preferably inhibits, reduces, disrupts, disassembles tau fibrilformation, deposition, accumulation and/or persistence, or treats adisease associated with these conditions, such as a tauopathy, by atleast 20%, more preferably by at least 40%, even more preferably by atleast 60%, and still more preferably by at least 80%, relative to anuntreated subject. Effective amounts of a compound of this invention orcomposition thereof for treatment of a mammalian subject are about 0.1to about 1000 mg/Kg of body weight of the subject/day, such as fromabout 1 to about 100 mg/Kg/day, especially from about 10 to about 100mg/Kg/day. A broad range of disclosed composition dosages are believedto be both safe and effective.

“Amyloid diseases” or “amyloidosis” suitable for treatment with thecompositions of this inventions are diseases associated with theformation, deposition, accumulation, and/or persistence of amyloidfibrils, especially the fibrils of an amyloid protein selected from thegroup consisting of beta-amyloid protein or Aβ, AA amyloid, AL amyloid,IAPP amyloid, PrP amyloid, α2-microglobilin amyloid, transthyretin,prealbumin, and procalcitonin, especially Aβ and IAPP amyloid, Suitablesuch diseases include Alzheimer's disease, Down's syndrome, Mildcognitive impairment (MCI), Cognitive Canine Dysfunction (CDD),traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE),concussions, hear trauma, single- and multiple blows to the head,post-traumatic stress disorders, dementia pugilistica, multiple systematrophy, inclusion body myositosis, hereditary cerebral hemorrhage withamyloidosis of the Dutch type, Niewman-Pick disease type C, cerebralβ-amyloid angiopathy, dementia associated with cortical basaldegeneration, the amyloidosis of type 2 diabetes, the amyloidosis ofchronic inflammation, the amyloidosis of malignancy and FamilialMediterranean Fever, the amyloidosis of multiple myeloma and B-celldyscrasias, the amyloidosis of prion diseases, Creutzfeldt-Jakobdisease, Gertsmann-Straussler syndrome, kuru, scrapie, the amyloidosisassociated with carpal tunnel syndrome, senile cardiac amyloidosis,familial amyloidotic polyneuropathy, and the amyloidosis associated withendocrine tumors.

“Fibrillogenesis” refers to the formation, deposition, accumulationand/or persistence of tau fibrils, filaments, inclusions, deposits,inclusions, or the like.

“Inhibition of fibrillogenesis” refers to the inhibition of formation,deposition, accumulation and/or persistence of such amyloid “plaque” ortau “tangle” fibril-like deposits.

“Disruption of fibrils or fibrillogenesis” refers to the disruption ofpreformed beta-amyloid or tau fibrils that usually exist in apre-dominant β-sheet secondary structure. Such disruption by compoundsof the invention may involve marked reduction or disassembly ofbeta-amyloid or tau fibrils as assessed by various methods such ascircular dichroism spectroscopy, Thioflavin S fluorometry,SDS-PAGE/Western blotting, or negative stain electron microscopy, asdemonstrated by the Examples presented in this application.

“Mammal” includes both humans and non-human mammals, such as companionanimals (dogs, cats and the like), laboratory animals (such as mice,rats, guinea pigs, and the like) and farm animals (cattle, horses,sheep, goats, swine, and the like).

“Plaques” refer to the meatball looking “amyloid deposits” consisting ofbeta-amyloid protein or Aβ found in various regions of brain (includinghippocampus, cerebral cortex, frontal cortex etc.) that is apathological hallmark of brain aging, mild-cognitive impairment (MCI),Alzheimer's disease, and is found in aging mammals such as dogs, cats(referred to as Cognitive Canine Dysfunction or CDD), monkeys, polarbears, horses and the like. Accumulation of amyloid “plaques” in brainis believed to lead to neurodegeneration, loss of synapses andconnections between neurons, cognitive decline, memory decline and loss,and loss of focus and concentration.

“Tangles” refer to the “dried-up” spaghetti looking “tangle-deposits”consisting of tau protein found in various regions of brain (includinghippocampus, cerebral cortex, frontal cortex etc.) that is apathological hallmark of brain aging, mild-cognitive impairment,Alzheimer's disease, concussions, traumatic brain injury (TBI), singleand repeated blows to the head, post-traumatic stress disorders, chronictraumatic encephalopathy (CTE) and the like.

“Tauopathies” suitable for treatment with the compounds of thisinvention are also diseases associated with the formation, deposition,accumulation, or persistence of tau fibrils. Suitable diseases includeAlzheimer's disease, frontotemporal lobar degeneration with tauinclusions (FTLD-tau) such as Pick's disease, progressive supranuclearpalsy, and corticobasal degeneration, agyrophillic grain disease, someprion diseases, amyotrophic lateral sclerosis/parkinsonism-dementiacomplex of Guam (also called Lytico Bodig disease), dementiapugilistica, chronic traumatic encephalopathy, Parkinson's disease andparticularly some genetic forms of Parkinson's disease,tangle-predominant dementia (with neurofibrillary tangles similar toAlzheimer's disease, but without amyloid plaques).

Tau fibrils is a generic term referring to a group of diverse, butspecific intracellular or extracellular protein deposits which all havecommon morphological properties, staining characteristics, and x-raydiffraction spectra.

“Treating” or “treatment” of a disease includes preventing the diseasefrom occurring in a mammal that may be predisposed to the disease butdoes not yet experience or exhibit symptoms of the disease (prophylactictreatment). Treatment can also mean inhibiting the disease (slowing orarresting its development), providing relief from the symptoms orside-effects of the disease (including palliative treatment), andrelieving the disease (causing regression of the disease), such as bydisruption of preformed tau fibrils. Treatment does not need beabsolute. One such preventive treatment may be use of the disclosedcompounds for the treatment of Mild Cognitive impairment (MCI).

The plant Uncaria tomentosa, also known as “Uña de Gato” (in Spanish) or“Cat's claw” (in English) refers to a woody vine which grows within thePeruvian Amazon rain forest. This slow-growing vine takes 20 years toreach maturity, and can grow over 100 feet in length as it attaches andwraps itself around the native trees. It is found abundantly in thefoothills, at elevations of two to eight thousand feet. The vine isreferred to as “cat's claw” because of its distinctive curved claw-likethorns that project from the base of its leaves. Uncaria tomentosa isexpected to have immune-supporting, anti-inflammatory, anti-viral,anti-mutagenic and antioxidant properties. The anti-inflammatoryproperties, for example, are expected to be beneficial for the treatmentof arthritis, rheumatism, bursitis and gout. Without being bound bytheory, it is believed that is beneficial effects in treating arthritispain can be due, in part, to its ability to cleanse the digestive tractand aid in removing toxins from the body. Furthermore, Uncaria tomentosaor cat's claw is expected to alleviate pain, and is expected to behelpful in reducing pain associated with, for example, chemotherapy,radiation treatment and AZT use.

Uncaria tomentosa or cat's claw is also expected to be useful instopping viral infections in stopping viral infections in the earlystages, fighting opportunistic infections in AIDs patients anddecreasing the viable size of some skin tumors and cysts. Uncariatomentosa can also be used to treat a variety of ailments, includingcancer, AIDs, Crohn's disease, respiratory infections, allergies,herpes, prostrate problems, lupus, Epstein Barr virus, chronic fatiguesyndrome, and a variety of stomach and bowl disorders.

For additional and further information and background on Uncariatomentosa, the reader is also referred to the inventors' WIPOInternational publication number WO98/51302, which is incorporatedherein by reference in its entirety.

Further Aspects and Utilizations of the Invention

Another embodiment of the present invention is to formulate, prior toadministration in a patient, a pharmaceutical blend comprising Uncariatomentosa and an oolong tea extract in one or more pharmaceuticalacceptable carriers, diluents or excipients.

In another embodiment Uncaria tomentosa obtained commercially in anyform could be further modulated using suitable carriers, excipients anddiluents including lactose, dextrose, sucrose, sorbitol, mannitol,starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin,calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone,cellulose, water syrup, methyl cellulose, methyl andpropylhydroxybenzoates, talc, magnesium stearate and mineral oil. Theformulations can additionally include lubricating agents, wettingagents, emulsifying and suspending agents, preserving agents, sweeteningagents or flavoring agents. The compositions of the invention may beformulated so as to provide quick, sustained or delayed response of theactive ingredient after administration to the patient. The compositionsare preferably formulated in a unit dosage form, each dosage containingfrom about 1 to about 10,000 mg of Uncaria tomentosa (or its activeingredients), more usually about 500 to about 2,000 mg of Uncariatomentosa (or its active ingredients).

However, it will be understood that the therapeutic dosage administeredwill be determined by the physician in the light of the relevantcircumstances including the clinical condition to be treated, the organor tissues affected or suspected to be affected with tau fibrilaccumulation, and the chosen route of administration. Therefore, theabove dosage ranges are not intended to limit the scope of the inventionin any way.

The term “unit dosage form” refers to physically discrete units suitableas unitary dosages for human subjects and other mammals, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect, in association with a suitablepharmaceutical carrier.

Use of extracts from the inner bark and root parts of Uncaria tomentosa,and its blends benefit human patients with tauopathies due to the newlydiscovered ability of Uncaria tomentosa in combination with an Oolongtea extract to inhibit tau fibril formation, inhibit tau fibril growth,inhibit tau fibril-proteoglycan interactions, inhibit taufibril-glycosaminoglycan interactions, and cause dissolution and/ordisruption of preformed tau fibrils.

Compositions and Administration

In general, isolated, and/or purified Uncaria tomentosa and oolong teaplant extracts are administered in therapeutically effective amounts byany of the usual modes known in the art, either singly or in combinationwith at least one of the extracts of the disclosure. Administration willbe by one of the following routes: oral, topical, and synthetic (e.g.transdermal, intranasal or by suppository), or parenteral (e.g.intramuscular, subcutaneous, or intravenous injection). Compositions cantake the form of tablets, pills, capsules, semisolids, powders,sustained release formulations, solutions, suspensions, elixers,aerosals, or any other appropriate compositions, and comprise at leastone pharmaceutically acceptable excipient. Suitable excipients are wellknown to persons of ordinary skill in the art, and they, and the methodsof formulating the compositions, can be found in standard references asAlfonso A R: Remington's Pharmaceutical Sciences, 17^(th) ed., MackPublishing Company, Easton, Pa., 1985. Suitable liquid carriers,especially for injectable solutions, include water, aqueous salinesolution, aqueous dextrose solution, and glycols.

In particular, the compound(s) can be administered orally, for example,as tablets, trouches, lozenges, aqueous or oily suspensions, dispersiblepowders or granules, emulsions, hard or soft capsules, syrups orelixers. In one embodiment, only one such compound is administered inany particular dosage form. Compositions intended for oral use can beprepared to any method known in the art for the manufacture ofnutraceutical compositions and such compositions can contain one or moreagents selected from the group consisting of sweetening agents,flavoring agents, coloring agents and preserving agents in order toprovide nutraceutically elegant and palatable preparations.

Tablets contain the plant extracts in admixture with non-toxicpharmaceutically acceptable excipients that are suitable for themanufacture of tablets. These excipients include, for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, maize starch or alginic acid; binding agents, for example,maize starch, gelatin or acacia; and lubricating agents, for examplemagnesium stearate or stearic acid or talc. The tablets can be uncoatedor they can be coated by known techniques to delay disintegration andabsorption in the gastrointestinal tract and thereby provide a sustainedaction over a longer period. For example, a time delay material such asglycerol monostearate or glycerol distearate can be employed.Formulations for oral use can also be prepared as hard gelatin capsuleswherein the compounds are mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules, wherein the active ingredient is mixed with water oran oil medium, for example, peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the compound in admixture with excipientssuitable for the manufacture of aqueous suspensions. Such excipientsinclude, for example, suspending agents, for example, sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethyl cellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;and dispersing and wetting agents that are naturally occurringphosphatides, for example lecithin, or condensation products of analkylene oxide with fatty acids; for example polyoxyethylene stearate,or condensation products of ethylene oxide with long chain aliphaticalcohols, for example heptadecaethyleneoxycetanol, or condensationproducts of ethylene oxide with partial esters derived from fatty acidssuch as hexitol, for example polyoxyethylene sorbitol monooleate, orcondensation products of ethylene oxide with partial esters from fattyacids and a hexitol annyhydride, for example polyethylene sorbitanmonooleate. The aqueous suspensions can also contain one or morepreservatives, for example ethyl or n-propyl p-hydroxybenzoate, one ormore coloring agents, one or more flavoring agents, and/or one or moresweetening agents, such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the extracts in avegetable oil, for example arachs oil, olive oil, sesame oil, or coconutoil, or in a mineral oil such as liquid paraffin. The oily suspensionscan contain a thickening agent for example beeswax, hand paraffin orcetyl alcohol. Sweetening agents, such as those set forth below, andflavoring agents can be added to provide a palatable oral preparation.These compositions can be preserved by the addition of an antioxidantsuch as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredients inadmixture with a dispersing or wetting agent, a suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already described above.Additional excipients, for example sweetening, flavoring and agents, canalso be present.

The plant extracts can also be in the form of oil-in-water emulsions.The oily phase of a vegetable oil, for example, olive oil or arachisoils, or a mineral oil, for example liquid paraffin, or mixturesthereof. Suitable emulsifying agents can be naturally-occurring gums,for example gum acacia or gum tragacanth, naturally occurringphosphatides, for example soy bean, lecithin, and esters or partialesters derived from fatty acids and hexitol anhydrides, for examplesorbitan esters with ethylene oxide, for example polyoxyethylenesorbitan monooleate, and condensation products of the said partialesters with ethylene oxide, for example polyethylene sorbitanmonooleate. The emulsion can also contain sweetening and flavoringagents. Syrups and elixers can be formulated with sweetening agents, forexample, glycerol, sorbitol or sucrose. Such formulations can alsocontain a demulcent, a preservative and flavoring and coloring agents.

The following non-limiting Examples are given by way of illustrationonly and are not considered a limitation of this disclosure, manyapparent variations of which are possible without departing from thespirit or scope thereof.

EXAMPLES Composition Preparation

For these studies, commercially available tea leaves or extracts wereobtained from various vendors. To make tea extracts from tea leaves, 2 gof tea leaves were extracted into 100° C. deionized water for 20 minwith occasional mixing. Tea extracts were filtered through a >10 μmcut-off filter to remove large particulates. Extracts were snap frozenin dry ice/ethanol then lyophilized to obtain a dried, concentratedpowder. Dried powder was weighed and resuspended in DMSO to makeconcentrated 100 mg/ml stock solutions. Stock solutions were dilutedinto aggregation reactions such that DMSO concentration was less than0.28% in the final reaction.

LOTE was prepared from oolong tea (AuNutra Industries Inc., Chino,Calif.). PTI-00703® is a powder extract made from water extraction ofthe inner bark of Peruvian Uncaria tomentosa (RFI Ingredients, Blauvelt,N.Y.). Methods for preparing PTI-00703® have been described, for examplein WIPO International Pub. No. WO98/51302. Ethanol extracts of oolongtea and/or Uncaria tomentosa are within the scope of the currentinvention.

Example 1: Compositions of this Invention are PotentDisrupters/Inhibitors of Alzheimer's Aβ Fibrils or Aggregates

The compositions were found to be potent disrupter/inhibitors of Aβprotein fibrils or aggregates, In a set of studies, the efficacy of thecompositions to cause disassembly/disruption/disaggregation ofpre-formed amyloid fibrils of Alzheimer's disease (i.e. consisting of Aβ1-42 or Aβ 1-40) was analyzed.

Part A: Thioflavin T Fluorometry

In this study, Thioflavin T fluorometry was used to determine theeffects of the compositions. Thioflavin T specifically binds tofibrillary amyloid, and this binding produces a fluorescence enhancementat 485 nm that is directly proportional to the amount of amyloid fibrilsformed. The higher the fluorescence, the greater the amount of amyloidfibrils formed (Nakai et al., Lab. Invest. 65:104-110, 1991; Levine III,Protein Sci. 2:404-410, 1993; Amyloid:Int. J. Exp. Clin. Invest. 2:1-6,1995).

In this study, 40 μl of a 1 mg/ml solution (in distilled water) orpre-fibrillized human Aβ 1-42 (rPeptide) was incubated at 37° C. for 3days either alone (control), or in the presence of LOTE+PTI-00703® (attest composition: Aβ weight ratios of 1:1 and 1:0.1) (referred to as“Cognitive Clarity™”). The final concentration of Aβ in the reaction was0.4 mg/ml (88 μM) in phosphate-buffered saline (PBS), pH 7.4+0.02%sodium azide in 100 μl final volume. Following 3-days of co-incubation,12.5 μl of each incubation mixture was transferred into a 96-wellmicrotiter plate containing 37.5 μl of PBS and 200 μl of a Thioflavin Tsolution (i.e. 125 μM Thioflavin T in 62.5 mM phosphate buffer, pH 6.8).The emission fluorescence was read at 485 nm (444 nm excitationwavelength) using an ELISA plate fluorometer after subtraction withbuffer alone or composition alone, as blank.

The results of the 3-day incubations are presented in FIG. 1. Incubationof Aβ 1-42 with “Cognitive Clarity” (i.e. combination ofLOTE+PTI-00703®) caused a dose-dependent disruption

disassembly/disaggregation ofpreformed Aβ 1-42 fibrils. At a testcomposition:Aβ weight ratio of 0.1:1 LOTE+PTI-00703 inhibited fibrils62.8%. At equal weight equivalents (test composition:Aβ weight ratio of1:1) there was a 93.6% inhibition of Thioflavin T fluorescence. Thisstudy indicated that this unique combination is a potentdisruptor/inhibitor of Alzheimer's disease type Aβ fibrils, and exertsits effect in a dose-dependent manner.

Part B: Congo Red

In the Congo red binding assay the ability of a test composition toalter β-amyloid binding to Congo red is quantified. In this assay, Aβ1-42 (as prepared for the Thioflavin T assay) was incubated for 3 dayseither alone (control), or with increasing amounts of test compositionsand then vacuum filtered through a 0.2 μm filter. The amount of Aβ1-42retained in the filter was then quantitated following staining of thefilter with Congo red (125 μM Congo red, 100 mM Tris, 50 mM NaCl, pH 7).After appropriate washing of the filter, any lowering of the Congo redcolor on the filter in the presence of the test composition (compared tothe Congo red staining of the amyloid protein in the absence of the testcomposition) was indicative of the test composition's ability todiminish/alter the amount of aggregated and congophilic Aβ.

In one study, the ability of Aβ fibrils to bind Congo red in the absenceor presence of increasing amounts of LOTE+PTI-00703® (at testcomposition:Aβ weight ratios of 1:1, and 0.1:1) was determined. Theresults of 3-day incubations are presented in FIG. 1. LOTE+-PTI-00703®caused a dose-dependent inhibition of Aβ binding to Congo red. At a testcomposition:Aβ weight ratio of 0.1:1, LOTE+PTI-00703® inhibited Congored binding 20.9% (p<0.001). At equal weight equivalents (testcomposition:Aβ weight ratio of 1:1) there was a 58.6% (p<0.001)inhibition of Congo red binding.

Similar to the results for the Congo red binding assay, this study alsoindicated that this combination of a specific lead oolong tea extract(LOTE) and PTI-00703® are potent disruptors/inhibitors of Aβ fibrils asassessed by a Thioflavin T fluorometry assay, and exerted its effects ina dose-dependent manner (FIG. 1). The combination of LOTE+PTI-00703®caused a dose dependent reduction of Thioflavin T binding (indicatingdisruption/reduction of Aβ fibrils) by 70% (p<0.001) at a testcomposition: Aβ weight ratio of 0.1:1 (FIG. 1), and by 95% at a testcomposition:Aβ weight ratio of 1:1 (FIG. 1).

Part C: Slide-Based Congo Red Binding, Thioflavin S and ElectronMicroscopy

In the slide-based Congo red assay, Congo red dye is incubated withAβ1-42, dotted on a slide and imaged under polarized light. Amyloidfibrils bound by Congo red emits a characteristic “apple-greenbirefringence” under polarized light. In this study, pre-fibrillarized0.4 mg/ml Aβ1-42 (as prepared for the Thioflavin T assay) with orwithout test compositions was incubated for 3 days. 10 μl Congo redsolution (250 mg Congo red dye (54% pure; Sigma) dissolved in 1 L dH20)was added to 10 μl of Aβ 1-42−/+test compositions and mixed by vortexingfor 30 seconds. Samples were incubated 10 minutes at room temperaturewith Congo red solution. Samples were then centrifuged at 2000 g for 3minutes and 10 μl of supernatant was removed. 2 μl glycerol was added tothe pellet and mixed by pipetting up and down 15 times. Sample wasvortexed then 10 μl of stained protein was dotted onto 18-well 5MMHTC(R) autoclavable blue slides. Samples were covered with smallcircular coverslips and then immediately imaged under polarized light.Images were captured with a Zeiss Axioscope 2 Plus microscope with HBO100 illuminator equipped with a Q-Imaging Retiga 1300 digital camera.

In FIG. 2A (left panel), a representative image shows that untreatedAβ1-42 has characteristic apple-green birefringence and abundantfibrillar protein uniformly distributed across the viewing field. InFIG. 2A (right panel) treatment with LOTE+PTI-00703 (0.1:1 weight ratiowith Aβ42) resulted in substantially less Congo red stained fibrils,demonstrating that the plant extract combination can reduce anddisaggregate/dissolve pre-formed Aβ 1-42 pre-formed fibrils.

Similar to Thioflavin T, Thioflavin S is a related anionic dye thatbinds to fibrillar amyloid proteins and can be used to detect fibrillarproteins bound to a glass microscope slide. In this study,pre-fibrillarized Aβ1-42 (as prepared for the Thioflavin T assay) withor without test compositions was incubated for 3 days. 4 μl of 0.4 mg/mlAβ 1-42−/+test compositions were dotted onto 18-well 5MM HTC(R)autoclavable blue slides. Samples were allowed to air dry for 2 h. 10 μlof Thioflavin S solution (31 mg of Thioflavin S dissolved in 50 mL dH20)was gently applied to dried protein on slides. Protein was stained for 1minute, then Thioflavin solution was removed by pipette. 40 μl of a 70%ethanol solution was gently pipetted onto stained protein for 1 minuteto rinse. This solution was gently removed by pipette. 2 μl ofVectashield (Vector) mounting media was applied to the stained proteinand then covered with circular coverslips. Images were viewed underfluorescent light and images captured with a Zeiss Axioscope 2 Plusmicroscope with HBO 100 illuminator equipped with a Q-imaging Retiga1300 digital camera.

In FIG. 2B (left panel), a representative image of untreated Aβ1-42shows abundant Thioflavin S fluorescent fibrils uniformly distributedacross the viewing field. Preformed Aβ1-42 fibrils incubated withLOTE+PTI-00703® (at 0.1:1 weight ratio with Aβ1-42) for 72 hr showed asubstantial reduction in fluorescent fibrils (FIG. 2B, right panel).

In FIG. 2B (right panel), representative images show that withLOTE+PTI-00703 treatment, the Thioflavin S-fluorescent fibrilsdisappeared.

Negative stain electron microscopy (EM) analysis was used toindependently monitor the effectiveness of different compositions todisrupt pre-formed Aβ fibrils. In these experiments, pre-formed. Aβ 1-42fibrils (as prepared for the Thioflavin T assay) were incubated in theabsence (control) or presence of increasing concentrations of testcompositions. After the 3-day incubation, 10 μl samples were spottedonto grids, stained with 2% uranyl acetate, and visualized at 8,000× to30,000× magnifications with a JEOL 1010 transmission electronmicroscope.

In FIG. 2C (bottom panel), EM analysis confirmed formation of Aβ fibrilsin the absence of treatment (i.e. FIG. 2C, Control). Without treatment(2C, left panel), Aβ formed large clumped fibrils that uniformly coveredthe field. These samples were also tested by the Thioflavin T assay andconfirmed to be Thioflavin T-fluorescence positive fibrils. In thepresence of LOTE+PTI-00703® (FIG. 2C, right panel), the number ofclumped Aβ fibrils was significantly reduced and dissolved. Theseresults correlate well with the Thioflavin T fluorometry data thatshowed a reduction in Thioflavin T fluorescence with treatment and Congored binding data that showed a reduction in Congo red binding withtreatment. Using these independent methodologies, we have identified andvalidated the lead oolong tea extract (LOTE) in combination withPTI-00703® (i.e. LOTE+PTI-00703®) as potent Aβ disruptors/inhibitors.

Example 2: Compositions of this Invention Directly Inhibit/Disrupt theIn Vitro Conversion of Aβ to β-Sheet Containing Fibril Structures PartA: Thioflavin T Fluorometry

To test whether LOTE+PTI-00703® can inhibit the 3-sheet formation of Aβ,the same Thioflavin T assay as described in Example 1 was utilized, butwith Aβ 1-40 as a substrate instead. Similar to Aβ1-42, Aβ1-40 formsThioflavin T positive aggregates but requires >24 h incubation at 37° C.shaking to become fully fibrillarized. Since Aβ 1-40 is in anon-fibrillar state at the start of the assay, this protein can beaggregated in the presence of compositions to measure aggregationinhibition. Lyophilized human Aβ 1-40 (rPeptide) was dissolved to 1mg/mL (220 μM) in dH20. In separate test tubes, test composition stockswere prepared in PBS at various concentrations such that final reactionscontaining equal volumes of the test composition stocks and the Aβsolution would result in a final Aβ concentration of 0.5 mg/mL (110 μM)with test composition:Aβ weight ratios of 1:1, 0.5:1, 1:1, and 0.2:1.The reactions containing Aβ+test compositions (or Aβ+PBS as a controlfor Aβ aggregation) were then incubated for 2 days. The incubationmixtures were diluted 1:10 to 0.05 mg/ml. Aβ and 50 μL of each dilutedincubation mixture was transferred into a 96-well microtiter platecontaining 200 μL of Thioflavin T solution (i.e. 125 μM Thioflavin T in62.5 mM phosphate buffer, pH 6.8). The fluorescence was read at 485 nm(444 nm excitation wavelength) using an ELISA plate fluorometer aftersubtraction with PBS buffer alone or composition alone, as blank.

The results of this study presented in FIG. 3 indicated thatLOTE+PTI-00703® of this invention interfered with Aβ aggregation asindicated by its ability to prevent the formation of fibrils as assessedby Thioflavin T fluorometry. At a test composition:Aβ weight ratio of0.2:1 LOTE+PTI-00703® inhibited fibrils by 14.1% and at 0.5:1LOTE+PTI-00703® inhibited fibrils at 73% (p<0.001). At equal weightequivalents (test composition:Aβ weight ratio of 1:1) there was an 89.3%inhibition (p<0.001) of Thioflavin T fluorescence. This study indicatedthat LOTE+PTI-00703® is a potent inhibitor of β-sheet rich-Aβ fibrilformation as assessed by Thioflavin T fluorometry, and this combinationexerts its effect in a dose-dependent manner.

Part B: Congo Red

To test whether LOTE+PTI-00703® can inhibit β-sheet formation of Aβ, thesame Congo red assay as described in Example 1 was utilized, but with Aβ1-40 as a substrate instead. In this assay, Aβ1-40 (as prepared for theThio T assay) and test compositions were incubated for 2 days and thenvacuum filtered through a 0.2 μm filter. The amount of Aβ 1-40 retainedin the filter was then quantitated following staining of the filter withCongo red (125 μM Congo red, 100 mM Tris, 50 mM NaCl, pH 7). Afterappropriate washing of the filter, any lowering of the Congo red coloron the filter in the presence of the test composition (compared to theCongo red staining of the amyloid protein in the absence of the testcomposition) was indicative of the test composition's ability todiminish/alter the amount of aggregated and congophilic Aβ.

In one study, the ability of Aβ fibrils to bind Congo red in the absenceor presence of increasing amounts of LOTE-1+PTI-00703® (at testcomposition:Aβ weight ratios of 1:1, 05:1, and 0.2:1) was determined.The results of 2-day incubations are presented in FIG. 3.LOTE+PTI-00703® caused a dose-dependent inhibition of A13 binding toCongo red. At a test composition:Aβ weight ratio of 0.2:1 LOTE+PTI-00703inhibited Congo red binding 41.7% and 05:1 LOTE+PTI-00703® inhibitedCongo Red binding 62.5% (p<0.001). At equal weight equivalents (testcomposition:Aβ weight ratio of 1:1) there was an 83.3% inhibition(p<0.001) of Congo red binding. Similar to the results for theThioflavin T fluorometry assay, this study also indicated thatLOTE+PTI-00703® is a potent inhibitor of Aβ fibrils as assessed by Aβfibril binding to Congo red, and exerts its effect in a dose-dependentmanner.

Part C: CD Spectroscopy

CD spectroscopy was performed to determine LOTE+PTI-00703®'s potency toinhibit formation of Aβ 1-40 β-sheet secondary structure underaggregation-prone conditions. Since β-sheet structure is characteristicof Aβ fibrils, monitoring secondary structure of protein can provideadditional proof of a compositions' effectiveness at inhibitingaggregation. CD spectra of Aβ 1-40 samples withincreasing−/+concentrations of compositions were analyzed at 25° C. on aJASCO Model J-810 Spectropolarimeter. CD spectroscopy and ThioT assayswere analyzed in parallel from the same sample preparation in order tocorrelate the results from two independent assays.

In FIG. 4, the CD spectra of Aβ 1-40 treated with LOTE+PTI-00703®indicated a dose-dependent inhibition of β-sheet containing fibrils. Aminima at 218 nm indicates the presence of β-sheet structure. A positiveshift of ellipticity at 218 nm indicates less β-sheet structure. At atest composition:Aβ weight ratio of 0.2:1 LOTE+PTI-00703® demonstrated30.9% less β-sheet structure from untreated. 0.5:1 LOTE+PTI-00703®showed 53.9% less β-sheet structure than control. At equal weightequivalents (test composition:Aβ weight ratio of 1:1) there was 64.8%less β-sheet structure compared to control.

These data verified LOTE+PTI-00703® has a significant ability to inhibitabnormal assembly of Aβ into fibrillar, β-sheet assemblies and maintainsAβ1-40 in a less pathogenic form.

Use of Recombinant Tau Repeat Domain for In Vitro Screening of TauAggregation Inhibitors

During in vitro screening for identification of tau aggregationinhibitors, we found that under the same experimental conditions,formation of paired helical filaments (PHFs) from commercially-purchasedfull-length tau protein (Tau441; from rPeptide) was much slower (>11days; data not shown) than that from the tau repeat domain (TauRD;containing Q244-E372 of Tau441) 24 hr) (S. Barghorn et al, Methods MolBiol, 299: 35-51, 2005).

Because of the remarkably short turn-around time and common aggregationproperties, we used TauRD for in vitro screening to identify tauaggregation inhibitors in our current invention. Since the TauRD proteinis not commercially available, we produced our own protein for thisproject. A cDNA fragment coding for the human TauRD was cloned into abacterial expression vector and the construct was then expressed in E.coli. Bacterial clones demonstrating high levels of expression of TauRDwere then selected for protein purification. The recombinant TauRDprotein was then purified by heat-stability treatment and cationexchange chromatography as described with minor modifications (S.Barghorn et al, Methods Mol Biol, 299: 35-51, 2005). Using this method,we achieved a protein yield of 20 mg per liter of bacterial culture).Aggregation and PHF formation of purified TauRD were evaluated andvalidated by independent assays including Thioflavin S (ThioS)fluorometry, a dye that fluorescences after binding to fibrils (FIG.5D), circular dichroism (CD) spectroscopy, a method that detects changesin secondary structure of proteins (FIGS. 5B and 5C) and electronmicroscopy (FIGS. 5E-G). The results consistently demonstrate that TauRD(10 μM) is able to form ThioS-positive, β-sheet-containing PHFs whenincubated with equimolar heparin (Sigma-Aldrich, St. Louis, Mo.) at 37°C., with shaking at 800-1000 rpm for day.

In FIG. 5A, TauRD (15 kDa) protein purified from E. coli was evaluatedby SDS-PAGE/silver stain with typical purity of >95%. In FIG. 5B-C areexamples of CD spectroscopy of non-aggregated and aggregated TauRDproteins. TauRD aggregates were prepared in the presence of equimolar(10 μM) ratios of TauRD and heparin in 20 mM sodium phosphate buffer(pH7.4), incubated at 37° C. with shaking for 0-120 h. In the absence ofheparin, CD spectra of non-aggregated TauRD was random coil withellipticity minima at 195 nm (FIG. 5B). In the presence of heparin, CDspectra showed time-dependent conformational changes of TauRD proteinfrom random coil (minima at 195 nm) at time 0 to β-sheet (minima at 218nm) at 20-120 h (FIG. 5C). FIG. 5D is an example of TauRD aggregationmonitored by ThioS fluorometry over time. The results in FIG. 5D, showformation of ThioS-positive TauRD fibrils after 20 h incubation. In theabsence of heparin without fibril formation, tauRD ThioS signals were<200 arbitrary units (A.U.) of fluorescence at all time points. Taufibril formation was confirmed by negative stain EM in FIG. 5E-G.Representative images show formation of Tau fibrils after 48 hincubation with heparin. TauRD monomers at time 0 are shown in FIG. 5E(Bar=200 nm). Formation of TauRD fibrils at 48 hr are shown in FIGS.5F-G (Bar=50 nm). Both straight and paired helical filaments were found.

Example 3: Identification of Novel Tau Aggregation Inhibitors byThioflavin S Fluorometry Screening

A well-known method for measuring fibril formation is Thioflavin T(ThioT) fluorometry (H. Naiki et al, Lab. Invest. 65:104-110, 1991; H.Levine III, Protein Sci. 2:404-410, 1993; H. Levine III, Amyloid 2:1-6,1995; H. Naiki and K. Nakakuki, Lab. Invest. 74:374-383, 1996). ThioT isknown to bind to fibrillar proteins, and an increase in fluorescencecorrelates with an increase in fibril formation, whereas a decrease influorescence correlates with a decrease in fibrils due to disassemblyand/or disruption. We modified the assay by replacing ThioT withThioflavin S (ThioS), a related anionic dye with similar properties,because the latter has been shown to be more sensitive, and reproduciblefor quantifying Tau PHFs (data not shown; P. Friedhoff et al,Biochemistry, 37(28): 10223-30, 1998). ThioS fluorometry was employed toassess whether the above described mixed compositions were capable ofcausing a disassembly/disruption of fibrils.

Aggregated tau fibrils were prepared in the presence of equimolar ratiosof TauRD and heparin (10 μM each) in 20 mM sodium phosphate buffer,pH7.4. The reaction mixture was incubated at 37° C. with shaking(800-1000 rpm) for 24 h to 72 h. Test compositions were tested atvarying weight to weight concentrations with 0.14 mg/ml TauRD withheparin. The same reaction mixtures (+increasing concentrations of testcompositions) but without TauRD were also set up in parallel to serve asbackground controls. For all test compounds background ThioSfluorescence readings were very low, usually <5% of those of theTauRD-containing wells.

Following 24-72 h of co-incubation, 50 μl of each incubation mixture wastransferred into a black 96-well microtiter plate (Santa CruzBiotechnology, Inc., Dallas, Tex.) with 50 μl of phosphate bufferedsaline (PBS; Sigma-Aldrich, St. Louis, Mo.) and 25 μl of Thioflavin Ssolution (500 mM Thioflavin S; Sigma-Aldrich, St. Louis, Mo. in PBS, pH7.4). Using an ELISA plate fluorometer, fluorescence was read at 485 nm(444 nm excitation wavelength) after subtraction with buffer alone orcomposition alone, as blank.

Inhibition of tau aggregation by 50%, IC₅₀, was calculated using Prismversion 5 software (GraphPad Software) by nonlinear regression [(Log[inhibitor] vs. normalized response; variable slope)]. In initialscreenings, the test compounds demonstrated a broad range of activitiesfor inhibiting tau protein fibril formation: IC₅₀ values ranged from<0.14 mg/ml (less or equal to the concentration of tauRD in the assay)to infinity (i.e. no activity at all). The results suggested that theinhibitory activities were test subject specific. A few samples werealso tested for their ability to disrupt preformed tau fibrils(disruption assay).

Over 25 tea extracts were tested for inhibition of ThioS-positive tauaggregates. A majority of teas were oolong tea extracts. FIG. 6, is anexample of ThioS fluorescence of tau aggregated in the presence of a fewoolong tea extracts, chamomile tea and green tea. Increasingconcentrations of extracts were incubated with 0.14 mg/ml (10 μM) tauRDand equimolar heparin for 24-72 h, shaking at 1000 rpm, 37° C. The leadoolong tea extract (LOTE) inhibited tau aggregates more than otheroolong teas, chamomile tea or green tea extract. LOTE inhibited tauaggregation 50% (IC₅₀) with a ratio of 0.23:1, weight:weight, ofcomposition to tau. This experiment demonstrates LOTE has a superiorability over other compositions to inhibit abnormal assembly of tau intofibrillar, pathogenic assemblies.

After identifying a tea extract that has excellent tau aggregationinhibition, lead oolong tea extract (LOTE) was incubated alone or incombination with PTI-00703® in the tau aggregation inhibition assay. Acombination of specific oolong tea and PTI-00703® inhibitedThioS-positive tau aggregates more than LOTE or PTI-00703® alone(*significant by paired t-test, p<0.05) (FIG. 7). This combination oflead oolong tea extract and PTI-00703® is a potent inhibitor of tauaggregation/fibrillogenesis that could be used as a novel therapeuticfor tauopathies.

Example 4. Tau Aggregation Inhibition by Lead Oolong Tea ExtractConfirmed by Analysis of Protein Secondary Structure by CircularDichroism (CD) Spectroscopy

CD is a powerful method that provides dye-independent confirmation thatthe structure of a protein has changed. CD measures the differentialabsorption between left and right handed circularly polarized light.Proteins contain elements of asymmetry that exhibit distinct CD signalsmeasured in units of ellipticity. Thioflavin S fluorescent, tauaggregates have an ellipticity minima of 218 nm, which is characteristicof β-sheet containing proteins. Non-aggregated tau has an ellipticitywith a characteristic minima at 195 nm indicating random coil structure.CD spectroscopy was performed to determine each composition's potency toinhibit formation of β-sheet secondary structure in TauRD underaggregation-prone conditions. CD spectra were taken from samplescontaining+/−TauRD with increasing concentrations of compounds, andanalyzed at 25° C. on a JASCO Model J-810 Spectropolarimeter. CDspectroscopy and ThioS assays were analyzed in parallel from the samesample preparation in order to correlate the results from twoindependent assays.

In FIG. 8, tau was treated with lead oolong tea extract (LOTE) orchamomile tea extract for 48 h. Tau treated with LOTE isdose-dependently inhibited from converting to β-sheet containingfibrils. At the highest concentration of LOTE treatment, tau remains insoluble, random coil form with a minima ˜195 nm. Conversely, at thehighest concentration of treatment with chamomile tea extract, tau stillchanges to an aggregated, β-sheet structure similar to the untreatedcontrol. These data confirm LOTE has a significant ability over othercompositions to inhibit abnormal assembly of tau into fibrillar, β-sheetassemblies and maintains tau in a non-pathogenic soluble, random coilform.

Example 5: Inhibition of Tau Protein Fibrillogenesis and Disaggregationof Preformed Tau Fibrils by PTI-00703+LOTE Determined by Negative StainElectron Microscopy (EM)

EM analysis was used to independently monitor the effectiveness ofcompositions to inhibit tau fibrillogenesis. In these experiments, taufibrils were assembled by incubating equimolar ratios of TauRD proteinand heparin (10 μM each) in the absence (control) or presence ofincreasing concentrations of test compositions. After the 2-dayincubation, samples were spotted onto grids, stained with 2% uranylacetate, and visualized at 8,000× to 30,000× magnifications with a JEOL1010 transmission electron microscope.

In FIG. 9, left panel, EM analysis confirmed formation of tau fibrils inthe absence of treatment. Without treatment, tau formed a mixture ofpaired straight and helical filaments similar to those found in humantauopathies (V. M. Lee et al., Ann. Rev. Neurosci. 24:1121-159, 2001).These samples were also tested by the ThioS assay and CD and confirmedto be ThioS-fluorescence positive and β-sheet in structure. In thepresence of PTI-00703® and PTI-00703®+LOTE (FIG. 9, middle and rightpanel), tau fibrils become shortened and sparse, indicating aninhibition of tau fibril formation. Even less fibrils are apparent intau samples treated with both PTI-00703® and LOTE. These resultscorrelate well with ThioS fluorometry and CD analysis, which showed areduction in ThioS fluorescence and decrease in β-sheet structure. Usingthree independent methodologies, we have identified and validatedPTI-00703+lead oolong tea extract as a potent inhibitor of tauaggregation/fibrillogenesis.

Utilizing EM analysis, preformed tau fibrils were shown to rapidlydisaggregate in the presence of both PTI-00703® and LOTE. TauRD wasincubated with equimolar heparin to form fibrils as described in theprevious assay. TauRD was diluted with or without test compositions andincubated, shaking at 37° C. for various time points. At each time pointtauRD+/−test compounds was assayed for ThioS fluorescence and snapfrozen for EM analysis. In FIG. 10 (left panel) without treatment,pre-fibrillarized tau remained in long filaments. In the next panels,pre-fibrillarized tau was found to rapidly disaggregate in the presenceof PTI-00703®+LOTE. As early as 15 minutes incubation withPTI-00703®+LOTE, tau fibrils are shorter and sparser than withouttreatment. Disruption of tau fibrils was also confirmed by ThioS assay(data not shown). These data indicate the composition of PTI-00703®+LOTEnot only can inhibit tau aggregation but can disaggregate preformed taufibrils.

Example 6: Further In Vivo Testing of the PTI-00703®+LOTE forImprovement of Cognition and Memory

Further in vivo studies are used to test the PTI-00703®+LOTE plantextract combination for their effectiveness in the reduction of brain“plaque and tangle” load and improvement of cognition and memory. 40-60men and women are selected for a clinical study. Subjects haveage-associated memory impairment (AAMI), and expecting to have worseningsymptoms of memory loss within the 6-month study period, but are in goodgeneral health otherwise.

This study includes a placebo group, i.e. the subjects are divided intotwo groups, one of which receives the PTI-00703®+oolong tea extractcombination capsules (two 390 mg capsules containing thePTI-00703®+oolong tea extract in a 1:1 weight/weight combination with ameal; preferably lunch) of this disclosure, and the other receives aplacebo (two capsules containing capsules without the study productactive ingredients). The patients are benchmarked as to memory,cognition, focus, concentration, reasoning and other symptoms associatedwith mild-cognitive impairment (MCI). Subjects in the test groupsreceive a therapeutic dose of the combination study product extract orplacebo for 6 months, with analysis for short-term memory, cognition,focus and concentration to be examined at 0, 1, 3, and 6-months oftreatmen. Accurate records with regards to memory, focus andconcentration are kept as to the benchmarked symptoms in both groups andat the end of the study these results are compared. The results also arecompared between members of each group. In addition, the results foreach patient are compared to the symptoms reported by each patientbefore the study began. Activity of the combination PTI-00703®+oolongtea extract study product is illustrated by the attenuation of thetypical cognitive decline, decline in short-term memory, cognition,focus and concentration, and/or associated behavioral disruptionsassociated with age-associated memory impairment (AAMI)

All references and patent publications provided herein are herebyincorporated by reference in their entireties.

1.-24. (canceled)
 25. A composition comprising a therapeuticallyeffective amount of an Uncaria tomentosa extract in combination with anoolong tea extract formulated for oral administration.
 26. Thecomposition of claim 25, wherein composition is a pill, a tablet, acaplet, a soft gelatin capsule, a hard gelatin capsule, a lozenge, asachet, a cachet, a vegicap, liquid drops, an elixir, a suspension, anemulsion, a solutions, a syrups, or a tea bag.
 27. The composition ofclaim 25, wherein the Uncaria tomentosa extract and the oolong teaextract are in the form of a lyophilized powder.
 28. The composition ofclaim 25, wherein the composition contains 100-500 mg of the Uncariatomentosa extract and 100-500 mg of the oolong tea extract.
 29. Thecomposition of claim 25, wherein the composition is an aqueoussuspension, a dispersible powder or granules.
 30. The composition ofclaim 25, wherein the composition further comprises a dispersing agent,a wetting agent, or a suspending agent.
 31. The composition of claim 25,wherein the composition comprises an oily suspension or an oil-in-wateremulsion, wherein the oil phase comprises the therapeutically effectiveamount of an Uncaria tomentosa extract and the oolong tea extract. 32.The composition of claim 31, wherein the composition further comprisesan emulsifying agent.
 33. The composition of claim 25, wherein thecomposition is formulated for delayed release after oral administration.34. The composition of claim 33, wherein the composition is a tablet ora capsule.
 35. The composition of claim 34, wherein the tablet is acoated tablet that delays absorption in the gastrointestinal tract. 36.The composition of claim 33, comprising 100-500 mg of the Uncariatomentosa extract and 100-500 mg of the oolong tea extract.